Biosynthesis of isopentenyl pyrophosphate

ABSTRACT

Methods for synthesizing isopentenyl pyrophosphate are provided. A first method comprises introducing into a host microorganism a plurality of heterologous nucleic acid sequences, each coding for a different enzyme in the mevalonate pathway for producing isopentenyl pyrophosphate. A related method comprises introducing into a host microorganism an intermediate in the mevalonate pathway and at least one heterologous nucleic acid sequence, each sequence coding for an enzyme in the mevalonate pathway necessary for converting the intermediate into isopentenyl pyrophosphate. The invention also provides nucleic acid sequences, enzymes, expression vectors, and transformed host cells for carrying out the methods.

TECHNICAL FIELD

The present invention relates to the biosynthesis of isopentenylpyrophosphate (IPP) and isoprenoids derived therefrom. Moreparticularly, the invention relates to methods for biosynthesizingisopentenyl pyrophosphate, and to nucleic acid sequences, enzymes,expression vectors, and transformed host cells for carrying out themethods.

BACKGROUND

Isoprenoids are compounds derived from the five-carbon molecule,isopentenyl pyrophosphate. Investigators have identified over 29,000individual isoprenoid compounds, with new ones continuously beingdiscovered. Isoprenoids are often isolated from natural products, suchas plants and microorganisms, which use isopentenyl pyrophosphate as abasic building block to form relatively complex structures. Vital toliving organisms, isoprenoids serve to maintain cellular fluidity andelectron transport, as well as function as natural pesticides, to namejust a few of their roles in vivo. Furthermore, the pharmaceutical andchemical communities use isoprenoids as pharmaceuticals, nutriceuticals,flavoring agents, and agricultural pest control agents. Given theirimportance in biological systems and usefulness in a broad range ofapplications, isoprenoids have been the focus of much attention byscientists.

Conventional means for obtaining isoprenoids include extraction frombiological materials (e.g., plants, microbes, and animals) and partialor total organic synthesis in the laboratory. Such means, however, havegenerally proven to be unsatisfactory. For example, organic synthesis isusually complex since several steps are required to obtain the desiredproduct. Furthermore, these steps often involve the use of toxicsolvents, which require special handling and disposal. Extraction ofisoprenoids from biological materials may also require toxic solvents.In addition, extraction and purification methods usually provide a lowyield of the desired isoprenoid, as biological materials typicallycontain only small quantities of these compounds. Unfortunately, thedifficulty involved in obtaining relatively large amounts of isoprenoidshas limited their practical use. In fact, the lack of readily availablemethods by which to obtain certain isoprenoids has slowed down theprogression of drug candidates through clinical trials. Furthermore,once an isoprenoid drug candidate has passed the usual regulatoryscrutiny, the actual synthesis of the isoprenoid drug may not lenditself to a commercial scale.

As a solution to such problems, researchers have looked to biosyntheticproduction of isoprenoids. Some success has been obtained in theidentification and cloning of the genes involved in isoprenoidbiosynthesis. For example, U.S. Pat. No. 6,291,745 to Meyer et al.describes the production of limonene and other metabolites in plants.Although many of the genes involved in isoprenoid biosynthesis may beexpressed in functional form in Escherichia coli and othermicroorganisms, yields remain relatively low as a result of minimalamounts of precursors, namely isopentenyl pyrophosphate.

In an effort to address the lack of isopentenyl pyrophosphate, someinvestigators have attempted to increase isopentenyl pyrophosphateproduction. Croteau et al. describe in U.S. Pat. No. 6,190,895 thenucleic acid sequences that code for the expression of1-deoxyxylulose-5-phosphate synthase, an enzyme used in one biologicalpathway for the synthesis of isopentenyl pyrophosphate. Low yields ofisopentenyl pyrophosphate remain, however, since several more enzymesare needed to catalyze other steps in this isopentenyl pyrophosphatebiosynthetic pathway. Further, the reference does not address analternative pathway for isopentenyl pyrophosphate biosynthesis, namelythe mevalonate pathway.

Thus, the current invention is directed toward solving these and otherdisadvantages in the art by increasing the typically low yieldsassociated with conventional synthesis of isopentenyl pyrophosphate andisoprenoids. Specifically, the current invention is directed towardidentification of new methods for the synthesis of isopentenylpyrophosphate, as isopentenyl pyrophosphate represents the universalprecursor to isoprenoid synthesis.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to overcome theabove-mentioned disadvantages of the prior art by providing a method forsynthesizing isopentenyl pyrophosphate in a host microorganism,comprising the step of introducing into the host microorganism aplurality of heterologous nucleic acid sequences, each coding for adifferent enzyme in the mevalonate pathway for producing isopentenylpyrophosphate.

It is another object of the invention to provide such a method whereinthe plurality of heterologous nucleic acid sequences is contained in atleast one extrachromosomal expression vector.

It is still another object of the invention to provide such a methodwherein the isopentenyl pyrophosphate is further synthesized into anisoprenoid.

It is yet another object of the invention to provide such a methodwherein the isoprenoid is selected from the group consisting of amonoterpene, sesquiterpene, diterpene, sesterterpene, triterpene,tetraterpene, and a steroid.

It is a further object of the invention to provide such a method whereinthe plurality of heterologous nucleic acid sequences further comprises aDNA fragment coding for an enzyme capable of converting isopentenylpyrophosphate to dimethylallyl pyrophosphate.

It is still a further object of the invention to provide a methodwherein the host microorganism is a prokaryote.

It is an additional object of the invention to provide a method whereinthe prokaryote is Escherichia coli.

Is it still another object of the invention to provide a method forsynthesizing isopentenyl pyrophosphate in a host microorganism, whereinthe method comprises introducing into the host microorganism anintermediate in the mevalonate pathway and at least one heterologousnucleic acid sequence, each said sequence coding for an enzyme in themevalonate pathway necessary for converting the intermediate intoisopentenyl pyrophosphate.

It is still a further object of the invention to provide DNA fragments,expression vectors, and host cells for carrying out the methodsdescribed herein.

Additional objects, advantages, and novel features of the invention willbe set forth in part in the description that follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned through routine experimentation uponpractice of the invention.

In one embodiment, the invention provides a method for synthesizingisopentenyl pyrophosphate in a host microorganism. The method comprisesintroducing into a host microorganism a plurality of heterologousnucleic acid sequences, each coding for a different enzyme in themevalonate pathway for producing isopentenyl pyrophosphate. As will beappreciated by those skilled in the art, the mevalonate pathway involvessix enzymes. The pathway starts from acetyl-CoA, proceeds through theintermediate mevalonic acid, and results in isopentenyl pyrophosphate.Of course, additional nucleotide sequences coding for other genes may beintroduced as well. In particular, nucleotide sequences coding forenzymes necessary in the production of specific isoprenoids may beintroduced into the host microorganism, along with those coding forenzymes in the mevalonate pathway. Preferably, at least oneextrachromosomal expression vector will be used to introduce the desirednucleic acid sequence(s), although more than one (e.g., two) differentexpression vectors may be used. In addition, the desired nucleic acidsequence(s) may be incorporated into the host microorganism'schromosomal material.

In another embodiment, the invention provides a method for synthesizingisopentenyl pyrophosphate in a host microorganism by introducing intothe host microorganism an intermediate of the mevalonate pathway and oneor more heterologous nucleic acid sequences. The introduced sequence orsequences each code for an enzyme in the mevalonate pathway necessaryfor converting the intermediate into isopentenyl pyrophosphate. Thus,for example, if mevalonate is the introduced intermediate, the methodrequires introduction of nucleic acid sequences that code for theenzymes necessary to convert mevalonate into isopentenyl pyrophosphate,for example, the introduction of nucleic acid sequences coding for anenzyme that phosphorylates mevalonate to mevalonate 5-phosphate, anenzyme that converts mevalonate 5-phosphate to mevalonate5-pyrophosphate, and an enzyme that converts mevalonate 5-pyrophosphateto isopentenyl pyrophosphate. Of course, other intermediates in themevalonate pathway, along with the necessary nucleic acid sequences, maybe introduced as well.

Although any host microorganism, e.g., a prokaryote or eukaryote, may beemployed, it is preferred that a prokaryote such as Escherichia coli beused. Preferably, the host organism does not synthesize isopentenylpyrophosphate through the mevalonate pathway, but rather through thedeoxyxylulose-5 phosphate (DXP) pathway. In this way, side reactionsinvolving the intermediates of the mevalonate pathway are minimized,thereby enhancing the yield and efficiency of the present methods.

In another embodiment of the invention, DNA fragments, each coding foran enzyme in the mevalonate pathway, are provided in one or moreexpression vectors. Thus, for the mevalonate pathway, the DNA fragmentsinclude those that code for enzymes capable of: (a) condensing twomolecules of acetyl-CoA to acetoacetyl-CoA, preferably the nucleotidesequence of SEQ ID NO 1; (b) condensing acetoacetyl-CoA with acetyl-CoAto form HMG-CoA, preferably the nucleotide sequence of SEQ ID NO 2; (c)converting HMG-CoA to mevalonate, preferably the nucleotide sequence ofSEQ ID NO 3; (d) phosphorylating mevalonate to mevalonate 5-phosphate,preferably the nucleotide sequence of SEQ ID NO 4; (e) convertingmevalonate 5-phosphate to mevalonate 5-pyrophosphate, preferably thenucleotide sequence of SEQ ID NO 5; and (f) converting mevalonate5-pyrophosphate to isopentenyl pyrophosphate, preferably the nucleotidesequence of SEQ ID NO 6.

In yet another embodiment, the invention provides expression vectorscomprising the DNA fragments described above and elsewhere in theapplication, as well as host cells transformed with such expressionvectors. The DNA fragments, expression vectors, and host cellstransformed with the same expression vectors are useful in the presentmethods for synthesizing isopentenyl pyrophosphate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically illustrate the mevalonate pathway ofisopentenyl pyrophosphate synthesis, along with enzymes involved andnucleic acid sequences coding for such enzymes.

FIG. 2 is a graph illustrating the difference in the concentration oflycopene produced from natural levels of isopentenyl pyrophosphate innon-engineered Escherichia coli and from Escherichia coli engineered tooverproduce isopentenyl pyrophosphate from a partialmevalonate-isoprenoid pathway, at different concentrations of mevalonate(Mev).

FIG. 3 is a graph illustrating the difference in normalized lycopeneconcentration produced from natural levels of isopentenyl pyrophosphatein non-engineered Escherichia coli and from Escherichia coli engineeredto overproduce isopentenyl pyrophosphate from the completemevalonate-isoprenoid pathway.

FIG. 4 is a graph illustrating the difference in amorphadieneconcentration produced from natural levels of isopentenyl pyrophosphatein non-engineered Escherichia coli and from Escherichia coli engineeredto overproduce isopentenyl pyrophosphate from a partialmevatonate-isoprenoid pathway.

FIG. 5 is a gas chromatographic spectrum illustrating the production ofditerpene using ethyl acetate extracts from Escherichia coli engineeredto produce isoprenoids from the artificial, modified MBIS operon (apartial mevalonate-isoprenoid pathway), and expressing a casbenecyclase.

For reference, FIG. 6 is the mass spectrum of the isoprenoid casbene.

DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail, it is to be understoodthat, unless otherwise indicated, this invention is not limited toparticular sequences, expression vectors, enzymes, host microorganisms,or processes, as such may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to an “expressionvector” includes a single expression vector as well as a plurality ofexpression vectors, either the same (e.g., the same operon) ordifferent; reference to “microorganism” includes a single microorganismas well as a plurality of microorganisms; and the like.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terms “optional” or “optionally” as used herein mean that thesubsequently described feature or structure may or may not be present,or that the subsequently described event or circumstance may or may notoccur, and that the description includes instances where a particularfeature or structure is present and instances where the feature orstructure is absent, or instances where the event or circumstance occursand instances where it does not.

The terms “host microorganism” and “cell” are used interchangeablyherein to refer to a living biological cell that can be transformed viainsertion of an expression vector. Thus, a host organism or cell asdescribed herein may be a prokaryotic organism (e.g., an organism of thekingdom Eubacteria) or a eukaryotic cell. As will be appreciated by oneof ordinary skill in the art, a prokaryotic cell lacks a membrane-boundnucleus, while a eukaryotic cell has a membrane-bound nucleus. Apreferred prokaryotic cell is Escherichia coli. Preferred eukaryoticcells are those derived from fungal, insect, or mammalian cell lines.

The term “heterologous DNA” as used herein refers to a polymer ofnucleic acids wherein at least one of the following is true: (a) thesequence of nucleic acids is foreign to (i.e., not naturally found in) agiven host microorganism; (b) the sequence may be naturally found in agiven host microorganism, but in an unnatural (e.g. greater thanexpected) amount; or (c) the sequence of nucleic acids comprises two ormore subsequences that are not found in the same relationship to eachother in nature. For example, regarding instance (c), a heterologousnucleic acid sequence that is recombinantly produced will have two ormore sequences from unrelated genes arranged to make a new functionalnucleic acid. Specifically, the present invention describes theintroduction of an expression vector into a host microorganism, whereinthe expression vector contains a nucleic acid sequence coding for anenzyme that is not normally found in a host microorganism. Withreference to the host microorganism's genome, then, the nucleic acidsequence that codes for the enzyme is heterologous.

The term “mevalonate pathway” is used herein to refer to the pathwaythat converts acetyl-CoA to isopentenyl pyrophosphate through amevalonate intermediate.

The terms “expression vector” or “vector” refer to a compound and/orcomposition that transduces, transforms, or infects a hostmicroorganism, thereby causing the cell to express nucleic acids and/orproteins other than those native to the cell, or in a manner not nativeto the cell. An “expression vector” contains a sequence of nucleic acids(ordinarily RNA or DNA) to be expressed by the host microorganism.Optionally, the expression vector also comprises materials to aid inachieving entry of the nucleic acid into the host microorganism, such asa virus, liposome, protein coating, or the like. The expression vectorscontemplated for use in the present invention include those into which anucleic acid sequence can be inserted, along with any preferred orrequired operational elements. Further, the expression vector must beone that can be transferred into a host microorganism and replicatedtherein. Preferred expression vectors are plasmids, particularly thosewith restriction sites that have been well documented and that containthe operational elements preferred or required for transcription of thenucleic acid sequence. Such plasmids, as well as other expressionvectors, are well known to those of ordinary skill in the art.

The term “transduce” as used herein refers to the transfer of a sequenceof nucleic acids into a host microorganism or cell. Only when thesequence of nucleic acids becomes stably replicated by the cell does thehost microorganism or cell become “transformed.” As will be appreciatedby those of ordinary skill in the art, “transformation” may take placeeither by incorporation of the sequence of nucleic acids into thecellular genome, i.e., chromosomal integration, or by extrachromosomalintegration. In contrast, an expression vector, e.g., a virus, is“infective” when it transduces a host microorganism, replicates, and(without the benefit of any complementary virus or vector) spreadsprogeny expression vectors, e.g., viruses, of the same type as theoriginal transducing expression vector to other microorganisms, whereinthe progeny expression vectors possess the same ability to reproduce.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free of components that normally accompanyit in its native state.

As used herein, the terms “nucleic acid sequence,” “sequence of nucleicacids,” and variations thereof shall be generic topolydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide that is an N-glycoside of a purine or pyrimidine base,and to other polymers containing nonnucleotidic backbones, provided thatthe polymers contain nucleobases in a configuration that allows for basepairing and base stacking, as found in DNA and RNA. Thus, these termsinclude known types of nucleic acid sequence modifications, for example,substitution of one or more of the naturally occurring nucleotides withan analog; internucleotide modifications, such as, for example, thosewith uncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), with negatively charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), and withpositively charged linkages (e.g., aminoalklyphosphoramidates,aminoalkylphosphotriesters); those containing pendant moieties, such as,for example, proteins (including nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.); those with intercalators (e.g.,acridine, psoralen, etc.); and those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.). As used herein, thesymbols for nucleotides and polynucleotides are those recommended by theIUPAC-IUB Commission of Biochemical Nomenclature (Biochemistry 9:4022,1970).

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter) and asecond nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

In a first embodiment, the invention provides a method for synthesizingisopentenyl pyrophosphate, the fundamental building block ofisoprenoids, in a host microorganism.

Isopentenyl pyrophosphate is also known as “isopentenyl diphosphate” andis commonly abbreviated as “IPP.” The method comprises introducing intothe host microorganism a plurality of heterologous nucleic acidsequences each coding for a different enzyme in the mevalonate pathwayfor producing isopentenyl pyrophosphate. As stated previously, themevalonate pathway for producing isopentenyl pyrophosphate in livingorganisms begins with acetyl-CoA and involves a mevalonate intermediate.

In another method for synthesizing isopentenyl pyrophophate, anintermediate in the mevalonate pathway is introduced into the hostmicroorganism. Although any method for introducing the intermediate maybe used, it is preferred to add the intermediate to the culture mediumused to grow the host microorganism. In this way, the intermediate istransported, e.g., via passive diffusion, across the cellular membraneand into the host microorganism.

Either before or after the intermediate is introduced, nucleic acidsequence(s) are introduced that code for those enzymes of the mevalonatepathway necessary to convert the intermediate into isopentenylpyrophosphate. As will be appreciated by one of ordinary skill in theart, the conversion from the intermediate into isopentenyl pyrophosphatemay require one, two, three, or more steps. Although any of theintermediates, i.e., acetyl Co-A, acetoacetyl-CoA, HMG-CoA, mevalonate,mevalonate 5-phosphate, and mevalonate 5-diphosphate, may be used,introduction of DL-mevalonate is a particularly preferred intermediatewhen using this method in the production of isopentenyl pyrophosphate.Enantiomers of any of the intermediates, such as the bioactiveenantiomer D-mevalonate, may be used as well.

As shown in the schematic of FIGS. 1A and 1B, the mevalonate pathwaycomprises six steps and involves six intermediates. Initially, twomolecules of acetyl-coenzyme A (more commonly referred to as“acetyl-CoA”) are combined. Acetyl-CoA is produced naturally by the hostmicroorganism when it is in the presence of a suitable carbon source.For example, eukaryotic cells naturally synthesize acetyl-CoA fromcompounds derived from sugars and fats. An enzyme capable of condensingtwo molecules of acetyl-CoA to acetoacetyl-CoA is used in this firststep of synthesizing isopentenyl pyrophosphate via the mevalonatepathway.

Thus, any DNA fragment coding for an enzyme capable of carrying out thisstep may be used in the present method. Preferably, however, the DNAfragment codes for an acetoacetyl-CoA thiolase. Genes for such thiolasesare known to those of ordinary skill in the art and include, forexample, the genes of acetyl-CoA thiolase from Ralstonia eutrophus(Peoples et al. (1989), “Poly-β-Hydroxybutyrate Biosynthesis inAlcaligenes eutrophus H16” and “Characterization of the Genes Encodingβ-Ketothiolase and Acetoacetyl-CoA Reductase,” J. Biol. Chem. 264 (26);5293-15297); Saccharomyces cerevisiae (S. cerevfsiae) (Hiser et al.(1994), “ERG10 From Saccharomyces cerevisiae Encodes Acetoacetyl-CoAThiolase, ” J. Biol. Chem. 269 (50); 31383-31389); and Escherichia coli.It is particularly preferred, however, that the thiolase encoded by thenucleotide sequence of SEQ ID NO 1 be used in the present method.

The next step in the mevalonate pathway requires the condensation ofacetoacetyl-CoA, formed from the preceding step, with yet anothermolecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA).This step is catalyzed enzymatically using an enzyme that will condenseacetoacetyl-CoA with acetyl-CoA.

Although any DNA fragment that codes for an enzyme capable of carryingout this step may be used, it is preferred that the DNA fragment codefor an HMG-CoA synthase. Known genes for HMG-CoA synthases include,without limitation, the synthases from Blattella germanica(Martinez-Gonzalez et al. (1993), “3-Hydroxy-3-Methylglutaryl-Coenzyme-ASynthase from Blattella germanica. Cloning, Expression, DevelopmentalPattern and Tissue Expression, ” Eur. J. Biochem, 217(2), 691-699); andS. cerevisiae, and thus, are preferred. A particularly preferredsynthase is encoded by the nucleotide sequence of SEQ ID NO 2.

The third step converts HMG-CoA to mevalonate. As with the other steps,this conversion is enzymatically controlled.

According to the present method, a DNA fragment coding for an enzymethat is capable of converting HMG-CoA into mevalonate is included in theexpression vector. The HMG-CoA reductase genes from Sulfolobussolfataricus (Bochar (1997), “3-Hydroxy-3-Methylglutaryl-Coenzyme AReductase of Sulfolobus solfataricus: DNA Sequence, Phylogeny,Expression in Escherichia coli of the hmgA Gene, and Purification andKinetic Characterization of the Gene Product, ” J. Bacteriol. 179(11):3632-3638); Haloferax volcanii (Bischoff et al. (1996),“3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase from Haloferaxvolcanii: Purification, Characterization, and Expression in Escherichiacoli,” J. Bacteriol. 178(1):19-23); and S. cerevisiae (Basson et al.(1988), “Structural and Functional Conservation Between Yeast and Human3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases, the Rate-LimitingEnzyme of Sterol Biosynthesis,” Mol Cell Biol. 8(9):3797-808) are known,and are consequently preferred for the present methods. It isparticularly preferred, however, that the nucleotide sequence of SEQ IDNO 3 that encodes an HMG-CoA reductase be used in the present methods.

The nucleotide sequence defined by SEQ ID NO 3 that encodes an HMG-CoAreductase is a truncated version of the S. cerevisiae gene coding forHMG-CoA reductase, HMG1. The protein coded for by HMG1 is an integralmembrane protein located in the endoplasmic reticulum of S. cerevisiae;it consists of a membrane-spanning, regulatory domain in its N-terminalregion (amino acids 1-552) and a catalytically active domain in itsC-terminal region. (See Polakowski (1998), “Overexpression of aCytosolic Hydroxymethylglutaryl-CoA Reductase Leads to SqualeneAccumulation in Yeast,” Appl. Microbiol Biotechnol. 49:66-71.) Thenucleotide sequence defined by SEQ ID NO 3 comprises an artificial startcodon, followed by nucleotides 1660-3165 of the HMG1 sequence.Therefore, the nucleotide sequence defined by SEQ ID NO 3 codes for onlythe catalytically active portion of S. cervisiae HMG-CoA reductase.

The fourth step in the mevalonate pathway involves the enzymaticphosphorylation of mevalonate to form mevalonate 5-phosphate.

Although any DNA fragment coding for an enzyme capable of mevalonatephosphorylation may be used, it is preferred that a DNA fragment codingspecifically for mevalonate kinase be used. Genes for such kinases areknown to those of ordinary skill in the art and include, for example,the mevalonate kinase of S. cerevisiae (Oulmouden et al. (1991),“Nucleotide Sequence of the ERG12 Gene of Saccharomyces cerevisiaeEncoding Mevalonate Kinase,” Curr. Genet. 19(1): 9-14). A particularlypreferred sequence that codes for this particular kinase is identifiedin SEQ ID NO4.

The fifth step in the mevalonate pathway requires the addition of asecond phosphate group to mevalonate 5-phosphate. An enzyme catalyzesthis step.

In the present method, a DNA fragment that codes for an enzyme capableof adding a second phosphate group to mevalonate 5-phosphate is used inthe expression vector. Preferably, the DNA fragment codes for aphosphomevalonate kinase, such as the gene of the same name obtainedfrom S. cerevisiae (Tsay et al. (1991), “Cloning and Characterization ofERG8, an Essential Gene of Saccharomyces cerevisiae that EncodesPhosphomevalonate Kinase,” Mol. Cell. Biol. 11(2):620-31). Such kinasesare known to those of ordinary skill in the art and include, forexample, the kinase coded by the nucleotide sequence of SEQ ID NO 5.

The sixth and final step of the mevalonate pathway is the enzymaticconversion of mevalonate 5-pyrophosphate into isopentenyl pyrophosphate.

Although any DNA fragment coding for a mevalonate pyrophosphatedecarboxylase may be used, it is particularly preferred that the genefrom S. cerevisiae (Toth et al. (1996), “Molecular Cloning andExpression of the cDNAs Encoding Human and Yeast MevalonatePyrophosphate Decarboxylase,” J. Biol. Chem. 271(14):7895-7898) be used.A particularly preferred DNA fragment is the nucleotide sequence of SEQID NO 6.

When an intermediate is introduced, the method additionally requiresintroduction of DNA fragments that code for enzymes responsible forcatalyzing those steps of the mevalonate pathway located “downstream”from the introduced intermediate. With reference to the mevalonatepathway described above and to the biosynthetic schemes provided inFIGS. 1A and 1B, one of ordinary skill in the art can readily determinewhich DNA fragments and enzymatic steps are necessary when a givenintermediate is introduced into the host microorganism.

The mevalonate pathway is contrasted with the mevalonate-independent (ordeoxyxylulose-5-phosphate) pathway. In some organisms, isopentenylpyrophosphate production proceeds by condensation of pyruvate andglyceraldehyde-3-phosphate, via 1-deoxyxylulose-5-phosphate (DXP) as anintermediate. (See Rohmer et al. (1993) Biochem. J. 295:517-524.) Whilesome organisms have genes for only one pathway, other organisms havegenes for both pathways. For a discussion of both the mevalonate anddeoxyxylulose 5-phosphate pathways, reference is made to Lange et al.(2000), “Isoprenoid Biosynthesis: The Evolution of Two Ancient andDistinct Pathways Across Genomes,” Proc. Natl. Acad. Sci. USA 97(24):13172-13177.

Any prokaryotic or eukaryotic host microorganism may be used in thepresent method so long as it remains viable after being transformed witha sequence of nucleic acids. Generally, although not necessarily, thehost microorganism is bacterial. Examples of bacterial hostmicroorganisms include, without limitation, those species assigned tothe Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoscilla, and Paracoccus taxonomical classes. Preferably,the host microorganism is not adversely affected by the transduction ofthe necessary nucleic acid sequences, the subsequent expression of theproteins (i.e., enzymes), or the resulting intermediates required forcarrying out the steps associated with the mevalonate pathway. Forexample, it is preferred that minimal “cross-talk” (i.e., interference)occur between the host microorganism's own metabolic processes and thoseprocesses involved with the mevalonate pathway

Those of ordinary skill in the art can readily identify suitable hostmicroorganisms. For example, cross-talk is minimized or eliminatedentirely when the host microorganism relies exclusively on the“deoxyxylulose 5-phosphate” (or “DXP”) pathway for synthesizingisopentenyl pyrophosphate. In such host microorganisms, the mevalonatepathway does not inherently influence (save for the additional synthesisof isopentenyl pyrophosphate) the host microorganism, since it lacks anygenes that are equipped to process the proteins (i.e., enzymes) orintermediates associated with the mevalonate pathway. Such organismsrelying exclusively or predominately on the deoxyxylulose 5-phosphatepathway include, for example, Escherichia coli. Of course, it will berecognized by those of ordinary skill in the art that the hostmicroorganism used in the method may also conduct isopentenylpyrophosphate synthesis via the mevalonate pathway, either exclusivelyor in combination with the deoxyxylulose 5-phosphate pathway.

Sequences of nucleic acids coding for the desired enzymes of themevalonate pathway are prepared by any suitable method known to those ofordinary skill in the art, including, for example, direct chemicalsynthesis or cloning. For direct chemical synthesis, formation of apolymer of nucleic acids typically involves sequential addition of3′-blocked and 5′-blocked nucleotide monomers to the terminal5′-hydroxyl group of a growing nucleotide chain; wherein each additionis effected by nucleophilic attack of the terminal 5′-hydroxyl group ofthe growing chain on the 3′-position of the added monomer, which istypically a phosphorus derivative, such as a phosphotriester,phosphoramidite, or the like. Such methodology is known to those ofordinary skill in the art and is described in the pertinent texts andliterature (e.g., in D. M. Matteuci et al. (1980) Tet. Lett. 521:719;U.S. Pat. No. 4,500,707 to Caruthers et al.; and U.S. Pat. Nos.5,436,327 and 5,700,637 to Southern et al.). In addition, the desiredsequences may be isolated from natural sources by splitting DNA usingappropriate restriction enzymes, separating the fragments using gelelectrophoresis, and thereafter, recovering the desired nucleic acidsequence from the gel via techniques known to those of ordinary skill inthe art, such as utilization of polymerase chain reactions. (See, forexample, U.S. Pat. No. 4,683,195 to Mullis.)

Once each of the individual nucleic acid sequences necessary forcarrying out the desired steps of the mevalonate pathway has beendetermined, each sequence must be incorporated into an expressionvector. Incorporation of the individual nucleic acid sequences may beaccomplished through known methods that include, for example, the use ofrestriction enzymes (such as BamHI, EcoRI, HhaI, XhoI, XmaI, and soforth) to cleave specific sites in the expression vector, e.g., plasmid.The restriction enzyme produces single stranded ends that may beannealed to a nucleic acid sequence having, or synthesized to have, aterminus with a sequence complementary to the ends of the cleavedexpression vector. Annealing is performed using an appropriate enzyme,e.g., DNA ligase. As will be appreciated by those of ordinary skill inthe art, both the expression vector and the desired nucleic acidsequence are often cleaved with the same restriction enzyme, therebyassuring that the ends of the expression vector and the ends of thenucleic acid sequence are complementary to each other. In addition, DNAlinkers may be used to facilitate linking of nucleic acids sequencesinto an expression vector.

A series of individual nucleic acid sequences can also be combined byutilizing methods that are known to those having ordinary skill in theart. (See, for example, U.S. Pat. No. 4,683,195 to Minshull et al.)

For example, each of the desired nucleic acid sequences can be initiallygenerated in a separate polymerase chain reaction (PCR). Thereafter,specific primers are designed such that the ends of the PCR productscontain complementary sequences. When the PCR products are mixed,denatured, and reannealed, the strands having the matching sequences attheir 3′ ends overlap and can act as primers for each other. Extensionof this overlap by DNA polymerase produces a molecule in which theoriginal sequences are “spliced” together. In this way, a series ofindividual nucleic acid sequences may be “spliced” together andsubsequently transduced into a host microorganism simultaneously. Thus,expression of each of the plurality of nucleic acid sequences iseffected.

Individual nucleic acid sequences, or “spliced” nucleic acid sequences,are then incorporated into an expression vector. The invention is notlimited with respect to the process by which the nucleic acid sequenceis incorporated into the expression vector. Those of ordinary skill inthe art are familiar with the necessary steps for incorporating anucleic acid sequence into an expression vector. A typicalexpression-vector contains the desired nucleic acid sequence preceded byone or more regulatory regions, along with a ribosome binding site,e.g., a nucleotide sequence that is 3-9 nucleotides in length andlocated 3-11 nucleotides upstream of the initiation codon in Escherchiacoli. See Shine et al. (1975) Nature 254.34 and Steitz, in BiologicalRegulation and Development: Gene Expression (ed. R. F. Goldberger), vol.1, p. 349, 1979, Plenum Publishing, N.Y., for discussions of ribosomebinding sites in Escherichia coli.

Regulatory regions include, for example, those regions that contain apromoter and an operator. A promoter is operably linked to the desirednucleic acid sequence, thereby initiating transcription of the nucleicacid sequence via an RNA polymerase enzyme. An operator is a sequence ofnucleic acids adjacent to the promoter, which contains a protein-bindingdomain where a repressor protein can bind. In the absence of a repressorprotein, transcription initiates through the promoter. When present, therepressor protein specific to the protein-binding domain of the operatorbinds to the operator, thereby inhibiting transcription. In this way,control of transcription is accomplished, based upon the particularregulatory regions used and the presence or absence of the correspondingrepressor protein. Examples include lactose promoters (LacI repressorprotein changes conformation when contacted with lactose, therebypreventing the LacI repressor protein from binding to the operator) andtryptophan promoters (when complexed with tryptophan, TrpR repressorprotein has a conformation that binds the operator; in the absence oftryptophan, the TrpR repressor protein has a conformation that does notbind to the operator). Another example includes the tac promoter. (SeedeBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25.) As will beappreciated by those of ordinary skill in the art, these and otherexpression vectors may be used in the present invention, and theinvention is not limited in this respect.

Although any suitable expression vector may be used to incorporate thedesired sequences, readily available expression vectors include, withoutlimitation: plasmids, such as pSC101, pBR322, pBBR1MCS-3, pUR, pEX,pMR100, pCR4, pBAD24, pUC19; bacteriophages, such as M 13 phage and λphage; as well as mutant phages, such as λgt-λβ. Of course, suchexpression vectors may only be suitable for a particular hostmicroorganism. One of ordinary skill in the art, however, can readilydetermine through routine experimentation whether any particularexpression vector is suited for any given host microorganism. Forexample, the expression vector can be introduced into the host organism,which is then monitored for viability and expression of the sequencescontained in the vector. In addition, reference may be made to therelevant texts and literature, which describe expression vectors andtheir suitability to any particular host microorganism.

The expression vectors of the invention must be introduced ortransferred into the host microorganism. Such methods for transferringthe expression vectors into host microorganisms are well known to thoseof ordinary skill in the art. For example, one method for transformingEscherchia coli with an expression vector involves a calcium chloridetreatment wherein the expression vector is introduced via a calciumprecipitate. Other salts, e.g., calcium phosphate, may also be usedfollowing a similar procedure. In addition, electroporation (i.e., theapplication of current to increase the permeability of cells to nucleicacid sequences) may be used to transfect the host microorganism. Also,microinjection of the nucleic acid sequencers) provides the ability totransfect host microorganisms. Other means, such as lipid complexes,liposomes, and dendrimers, may also be employed. Those of ordinary skillin the art can transfect a host microorganism with a desired sequenceusing these or other methods.

For identifying a transfected host microorganism, a variety of methodsare available. For example, a culture of potentially transfected hostmicroorganisms may be separated, using a suitable dilution, intoindividual cells and thereafter individually grown and tested forexpression of the desired nucleic acid sequence. In addition, whenplasmids are used, an often-used practice involves the selection ofcells based upon antimicrobial resistance that has been conferred bygenes intentionally contained within the expression vector, such as theamp, gpt, neo, and hyg genes.

The host microorganism is transformed with at least one expressionvector. When only a single expression vector is used (without theaddition of an intermediate), the vector will contain all of the nucleicacid sequences necessary for carrying out isopentenyl pyrophosphatesynthesis via the mevalonate pathway. Although such an all-encompassingexpression vector may be used when an intermediate is introduced, onlythose nucleic acid sequencers) necessary for converting the intermediateto isopentenyl pyrophosphate are required.

When two versions of an expression vector are used (without the additionof an intermediate), nucleic acid sequences coding for some of the sixproteins (i.e., enzymes) necessary for isopentenyl synthesis via themevalonate pathway may be contained in a first expression vector, whilethe remainder are contained in a second expression vector. Again, thenucleic acid sequence(s) necessary for converting an introducedintermediate into isopentenyl pyrophosphate will be contained in theexpression vector(s). As will be appreciated by those of ordinary skillin the art, a number of different arrangements are possible, and theinvention is not limited with respect to the particular arrangementused.

Once the host microorganism has been transformed with the expressionvector, the host microorganism is allowed to grow. For microbial hosts,this process entails culturing the cells in a suitable medium. It isimportant that the culture medium contain an excess carbon source, suchas a sugar (e.g., glucose) when an intermediate is not introduced. Inthis way, cellular production of acetyl-CoA, the starting materialnecessary for isopentenyl pyrophosphate production in the mevalonatepathway, is ensured. When added, the intermediate is present in anexcess amount in the culture medium.

As the host microorganism grows and/or multiplies, expression of theproteins (i.e., enzymes) necessary for carrying out the mevalonatepathway, or for carrying out one or more steps within the pathway, iseffected. Once expressed, the enzymes catalyze the steps necessary forcarrying out the steps of the mevalonate pathway, i.e., convertingacetyl-CoA into isopentenyl pyrophosphate. If an intermediate has beenintroduced, the expressed enzymes catalyze those steps necessary toconvert the intermediate into isopentenyl pyrophosphate. Any means forrecovering the isopentenyl pyrophosphate from the host microorganism maybe used. For example, the host microorganism may be harvested andsubjected to hypotonic conditions, thereby lysing the cells. The lysatemay then be centrifuged and the supernatant subjected to highperformance liquid chromatography (HPLC). Once the isopentenylpyrophosphate is recovered, modification may be carried out in thelaboratory to synthesize the desired isoprenoid.

If desired, the isopentenyl pyrophosphate may be left in the hostmicroorganism for further processing into the desired isoprenoid invivo. For example, large amounts of the isoprenoid lycopene are producedin Escherichia coli specially engineered with the expression vectorpAC-LYC, as shown in Examples 3 and 4. Lycopene can be recovered usingany art-known means, such as those discussed above with respect torecovering isopentenyl pyrophosphate. Lycopene is an antioxidantabundant in red tomatoes and may protect males from prostate cancer.(See Stahl et al. (1996) Ach. Biochem. Biophys. 336(1):1-9.) Of course,many other isoprenoids can be synthesized through other pathways, andthe invention is not limited with respect to the particular “downstream”pathway. Thus, the present method not only provides methods forproducing isopentenyl pyrophosphate, but offers methods for producingisoprenoids as well.

Optionally, when it is desired to retain isopentenyl pyrophosphate inthe host microorganism for further biochemical processing, it ispreferred that the heterologous nucleic acid sequences introduced intothe host microorganism also include a DNA fragment coding for an enzymecapable of converting isopentenyl pyrophosphate to dimethylallylpyrophosphate. As appreciated by those of ordinary skill in the art, asuitable isomerase will catalyze the conversion of isopentenylpyrophosphate into dimethylallyl pyrophosphate. Such isomerases areknown to those of ordinary skill and include, for example, theisopentenyl pyrophosphate isomerase (idi) coded by the nucleotidesequence of SEQ ID NO 10. Isoprenoid biosynthetic pathways requiredimethylallyl pyrophosphate, and increased expression of the isomeraseensures that the conversion of isopentenyl diphoshate into dimethylallylpyrophosphate does not represent a rate-limiting step in the overallpathway.

The present methods thus provide for the biosynthetic production ofisopentenyl pyrophosphate and isoprenoids derived therefrom. As statedabove, isopentenyl pyrophosphate has been available only in relativelysmall amounts, and the present methods provide a means for producingrelatively large amounts of this important compound.

Further, the invention provides the ability to synthesize increasedamounts of isoprenoids. As stated above, isoprenoids represent animportant class of compounds and include, for example, food and feedsupplements, flavor and odor compounds, and anticancer, antimalarial,antifungal, and antibacterial compounds. Preferred isoprenoids are thoseselected from the group consisting of monoterpenes, sesquiterpenes,diterpenes, sesterterpenes, triterpenes, tetraterpenes, and steroids. Asa class, terpenes are classified based on the number of isoprene unitscomprised in the compound. Monoterpenes comprise ten carbons or twoisoprene units, sesquiterpenes comprise 15 carbons or three isopreneunits, diterpenes comprise 20 carbons or four isoprene units,sesterterpenes comprise 25 carbons or five isoprene units, and to soforth. Steroids (generally comprising about 27 carbons) are the productsof cleaved or rearranged terpenes.

Monoterpenes include, for example, flavors such as limonene, fragrancessuch as citranellol, and compounds having anticancer activity, such asgeraniol. Sesquiterpenes include, without limitation: periplanone B, acockroach hormone used to lure cockroaches into traps; artemisinin, anantimalarial drug; ginkgolide B, a platelet-activating factorantagonist; forskolin, an inhibitor of adenylate cyclase; and farnesol,a compound shown to have anticancer activity. Nonlimiting examples ofditerpenes include the antibacterial and antifungal compound casbene andthe drug paclitaxel. Among triterpenes (C₃₀) and tetraterpenes (C₄₀) arecarotenoids, which are used as antioxidants, coloring agents in food andcosmetics, and nutritional supplements (e.g., as vitamin A precursors).As pathways to these and other isoprenoids are already known, theinvention can advantageously be incorporated into an overall scheme forproducing relatively large amounts of a desired isoprenoid.

Conveniently, the invention also provides sequences, enzymes, expressionvectors, and host cells or microorganisms for carrying out the presentmethods. For example, the six genes necessary for isopentenylpyrophosphate synthesis from acetyl-CoA are conveniently provided in SEQID NO 7. In addition, the invention also provides sequences for thefirst three genes and the last three genes in SEQ ID NOs 8 and 9,respectively. These sequences can easily be included in an expressionvector using techniques described herein or other techniques well knownto those of ordinary skill in the art. In addition, the invention alsoprovides host cells transformed with one or more of these expressionvectors for use in carrying out the present methods.

It is to be understood that, while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications withinthe scope of the invention will be apparent to those skilled in the artto which the invention pertains.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entireties.

Experimental

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of the biosynthetic industry and thelike, which are within the skill of the art. Such techniques areexplained fully in the literature.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.), butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees Celsius and pressure isat or near atmospheric pressure at sea level. All reagents, unlessotherwise indicated, were obtained commercially

EXAMPLE 1 Cloning of the Mevalonate Pathway Operons

Assembly of the Mevalonate Operons

Individual genes for a mevalonate-isoprenoid pathway were assembled toform artificial complete and at least one functional operon. Cloning ofthe nucleic acid sequences coding for the enzymes of the mevalonatepathway was carried out and the reproduced sequences were divided intotwo operons. In one of the two operons, the last three genes of thebiosynthetic pathway (mevalonate kinase (MK)—SEQ ID NO 4;phosphomevalonate kinase (PMK)—SEQ ID NO 5; and mevalonate pyrophosphatedecarboxylase (MTD)—SEQ ID NO 6) were cloned by a polymerase chainreaction (PCR) as one operon by splicing the genes together usingoverlap extensions (SOEing). This operon is referred to as themevalonate bottom (MevB) operon (SEQ ID NO 9). In the second of the twooperons, the first three genes of the pathway (acetoacetyl-CoA thiolase(atoB)—SEQ ID NO 1; HMG-CoA synthase (HMGS)—SEQ ID NO 2; and a truncatedversion of HMG-CoA reductase (tHMGR)—SEQ ID NO 3) were cloned as aseparate artificial operon using the same technique. This operon isreferred to as the mevalonate top (MevT) operon (SEQ ID NO 8). Theindividual genes were isolated by PCR from genomic DNA of Saccharomycescerevisiae and Escherichia Coli prepared by established microbiologicprotocols. (See Sambrook et al., Molecular Cloning: a Laboratory Manual,3rd ed., Cold Harbor Springs Laboratory Press.) The 100 μL PCR reactionscontained 1×Pfu buffer, 1.5 mM MgSO₄ (Stratagene, La Jolla, Calif.), 200μM of each dNTP (Gibco BRL™, Life Technologies, Inc., Gaithersburg,Md.), 500 μM of each primer, 100 to 500 ng of template DNA, 5% dimethylsulfoxide (Sigma, St. Louis, Mo.), and 2.5 U of Pfu Turbo DNA polymerase(Stratagene). The reactions were carried out in a PTC-200 PeltierThermal Cycler from MJ Research (South San Francisco, Calif.) with thefollowing temperature cycling program: an initial heating step up to 95°C. for four minutes was followed by 30 cycles of 30 seconds ofdenaturing at 95° C., 30 seconds of annealing at 50° C., and 100 secondsof extension at 72° C., followed by one cycle at 72° C. for ten minutes.Once each gene of the operon was amplified from genomic DNApreparations, the operons were assembled by PCR reactions similar to theprocedure described above, but using the amplified DNA of all threegenes as template DNA and only the forward primer of the outermost 5′gene and the reverse primer of the outermost 3′ gene. The assembledoperons were isolated on 0.7% agarose gels and purified using a Qiagengel purification kit (Valencia, Calif.) according to the manufacturer'sinstructions.

Cloning Mevalonate Operons into Sequencing and Expression Vectors

As expression of biochemical pathways is often suboptimal from high-copyplasmids containing strong promoters, the artificial mevalonateoperon(s) were cloned in a variety of expression vectors to determinethe effect of plasmid copy number and promoter strength on expression ofthe cloned pathway. Prior to testing for pathway expression, theassembled operons were cloned into the pCR4 TOPO vector using theInvitrogen TOPO TA cloning system (Carlsbad, Calif.) for sequencingpurposes. Ligation into pCR4 TOPO vector and transformation ofEscherichia coli TOP10 cells were carried out according to themanufacturer's instructions. The synthetic operons were excised from thesequenced pCR4 TOPO vectors using restriction enzymes and ligated intothe high-copy vector pBAD24, which contains the arabinose-induciblearaBAD promoter (Guzman et al. (1995) J. Bacteriology 177:4121-4130);pTrc99A, which contains the IPTG-inducible tac promoter (Amann et al.(1988) Gene 69:301-315); or into pBBR1MCS-3 (Kovach et al. (1995) Gene166:175-176) or pUC19 (Yanisch-Perron et al. (1985) Gene 33:103-119),which contain the unregulated Lac promoters (no plasmid-encoded LacI).The MevB operon was digested with PstI and ligated using T4 DNA ligase(New England Biolabs, Inc., Beverly, Mass.) into the PstI site of thelow-copy vector, pBBR1MCS-3, containing P_(Lac) promoter andtetracycline resistance marker. The resulting plasmid, which encodes theenzymes responsible for the conversion of mevalonate to isopentenylpyrophosphate, was named pBBRMevB. The MevT operon was cloned into theSalI site of pBAD24 by digesting with SalI restriction enzyme andligating with T4 DNA ligase. The resulting plasmid, which encodes theenzymes responsible for the conversion of acetyl-CoA to mevalonate, wasnamed pBADMevT.

Addition of Isopentenyl Pyrophosphate Isomerase to MevB Operon

The syntheses of geranyl pyrophosphate (GPP), farnesyl pyrophosphate(FPP), and geranylgeranyl pyrophosphate (GGPP) require both isopentenylpyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP),to create the backbone structure of all isoprenoids. To ensuresufficient production of DMAPP from IPP, an additional gene, idi(encoding isopentenyl pyrophosphate isomerase, SEQ ID NO 10), wasamplified by PCR from Escherichia coli genomic DNA using primerscontaining an XmaI restriction enzyme site at the 5′ ends. Both theamplified product (containing idi) and pBBRMevB were digested with XmaIand ligated, thereby placing idi at the 3′ end of the MevB artificialoperon. The resulting operon, containing the MevB operon and idi, isdesignated MBI (SEQ ID NO 12). The resulting plasmid (containing theoperon of genes that encode for enzymes that convert mevalonate to IPPand DMAPP) was named pBBRMBI-2.

Addition of Polyprenyl Pyrophosphate Synthase(s) to MBI Operon

In order to direct products of the mevalonate pathway operons to thedifferent classes of isoprenoids (monoterpenes, sesquiterpenes,diterpenes, etc.), various polyprenyl pyrophosphate synthases werecloned into the MBI operon, such as geranyl diphosposphate (GPP)synthase, farnesyl pyrophosphate (FPP) synthase, and geranylgeranylpyrophosphate (GGPP) synthase. Polyprenyl pyrophosphate synthases werecloned by PCR using forward primers with a SacII restriction site andreverse primers with a SacI restriction site. Using restriction enzymesand T4 DNA ligase, the polyprenyl pyrophosphate synthases were clonedbetween the SacII and SacI sites of pBBRMBI-2. For example, farnesylpyrophosphate synthase gene ispA (SEQ ID NO 11) was isolated by PCR fromEscherichia coli genomic DNA and cloned between the SacII and SacI sitesof pBBRMBI-2,3′ of idi and the MevB operon. The resulting operon,containing the MevB operon, idi, and ispA (SEQ ID NO 11) has beendesignated MBIS (SEQ ID NO 13). The plasmid, which encodes the enzymesresponsible for the synthesis of farnesyl pyrophosphate (FPP) frommevalonate, was named pBBRMBIS-2.

EXAMPLE 2 Functionality of the Engineered Mevalonate Operon(s) byGrowth/No-Growth Phenotype

Functionality of the various genetic constructs was shown by expressionof the artificial mevalonate-isoprenoid pathway. The plasmids wereintroduced into an Escherichia coli host in which themevalonate-independent (DXP) isoprenoid pathway was inactivated.Escherichia coli strain DMY1 (Kuzuyama et al. (1999) Biosci. Biotechnol.Biochem. 63:776-778) contains a mutation (insertion/deletion) in thegene encoding for 1-deoxyxylulose 5-phosphate reductoisomerase (or DXR,the second step of the DXP pathway) that causes inactivation of themevalonate-independent pathway. Since this mutation is lethal toEscherichia coli, the strain must be propagated in Luria-Bertoni (LB)medium (available from, for example, Sigma, St. Louis, Mo.) containing0.5 mM of methylerithrytol (ME), the product of DXR; or it must have analternate pathway for the production of isopentenyl pyrophosphate.

Cultures of Escherichia coli strain DMY1 were made electrocompetentaccording to the method of Sambrook et al. (above) and transformed withpBBRMBI-2, or both pBBRMBI-2 and pBADMevT. Newly transformed DMY1 cellswere first allowed to recover on LB agar plates overnight, and weresupplemented with 0.5 mM ME and appropriate antibiotics at 37° C. priorto testing growth on media devoid of ME. DMY1 cells transformed withonly pBBRMBI-2 were plated on LB agar devoid of ME, but supplementedwith 1 mM DL-mevalonate prepared by incubating 1 volume of 2 MD-mevalonic acid lactone (Sigma, St. Louis, Mo.) with 1.02 volumes of 2M KOH at 37° C. for 30 minutes. DMY1 cells transformed with bothpBBRMBI-2 and pBADMevT plasmids were plated on LB agar with antibioticsonly (no ME or DL-mevalonate). All test plates were incubated for 48hours at 37° C.

Escherichia coli strain DMY1 cells containing pBBRMBI-2 were able togrow on LB agar plates with 1 mM DL-mevalonate, whereas Escherichia coliDMY 1 cells without the plasmid or with pBBR1MCS-3 (empty vectorcontrol) did not grow. The MBI operon successfully converted thesupplemented mevalonate to isopentenyl pyrophosphate and dimethylallylpyrophosphate, thereby complementing the dxr deletion.

Escherichia coli strain DMY1 cells containing pBADMevT and pBBRMBI-2were able to grow on LB agar plates not supplemented with DL-mevalonate,whereas Escherichia coli DMY1 cells without either of the plasmids couldnot grow on LB agar alone. The expression of the MevT and MBI operonssuccessfully converted acetyl-CoA to isopentenyl pyrophosphate anddimethylallyl pyrophosphate in vivo, thereby restoring growth toEscherichia coli strain DMY1, in which the native DXP isoprenoid pathwayis inactive.

EXAMPLE 3 Production of Carotenoids from Mevalonate Using the MBIArtificial Operon

The production of a carotenoid was used to demonstrate the benefits ofexpressing the artificial mevalonate-dependent IPP biosynthetic pathwayover the native Escherichia coli DXP-isoprenoid pathway. The increasedproductivity of the mevalonate-dependent isopentenyl pyrophosphatebiosynthetic pathway encoded by the synthetic operons was assayed bycoupling isopentenyl pyrophosphate production to the production oflycopene. This was accomplished by co-transforming Escherichia coli withpAC-LYC, a low-copy broad-host plasmid that expresses the genes encodingthe pathway for lycopene production from farnesyl pyrophosphate. Thegenes expressed from pAC-LYC are crtE (geranylgeranyl pyrophosphatesynthase), crtB (phytoene synthase), and crtI (phytoene desaturase) fromErwinia herbicola, which were cloned into pACYC 184 using methodssimilar to those described in Examples 1 and 2. Escherichia colinaturally produces farnesyl pyrophosphate from two molecules ofisopentenyl pyrophosphate and one molecule of dimethylallylpyrophosphate through the enzyme farnesyl pyrophosphate synthase, ispA(SEQ ID NO 11). Alternatively, more flux can be directed from themevalonate pathway to the lycopene pathway by including the Escherichiacoli gene encoding farnesyl pyrophosphate synthase into the artificialoperon(s).

From previous experiments (not described herein), it was found that theproduction of isopentenyl pyrophosphate from the mevalonate pathwayoperons was greater in the Escherichia coli strain DH10B than in theEscherichia coli strain DMY1. In order to demonstrate isopentenylpyrophosphate production from the mevalonate pathway only, the geneencoding 1-deoxyxylulose 5-phosphate reductoisomerase, dxr, wasinactivated in Escherichia coli strain DH10B by the method detailed byDatsenko et al. (2000), “One-step Inactivation of Chromosomal Genes inEscherichia Coli K-12 Using PCR Products,” PNAS 97:6640-6645. In theresulting Escherichia coli strain, named DPDXR1, the mevalonateindependent pathway (or DXP pathway) is inactive, and in order tosurvive, the strain must either be propagated in LB medium containing0.5 mM of methylerithrytol (ME) or have an alternate pathway for theproduction of isopentenyl pyrophosphate.

Escherichia coli strain DPDXR1 was transformed with pAC-LYC andpBBRMBI-2, while Escherichia coli strain DH10B was transformed withpAC-LYC and pBBR1MCS-3 (control) by electroporation. Transformants wereselected on LB agar plates supplemented with 50 μg/ml chloramphenicol,10 μg/ml tetracycline, and 1 mM DL-mevalonate by incubating overnight at37° C. One colony of each strain (Escherichia coli DPDXR1 harboringpAC-LYC and pBBRMBI-2 or Escherichia coli DH10B harboring pAC-LYC andpBBR1MCS-3) was transferred from the LB agar selection plate to 5 ml ofLB liquid medium also supplemented with 50 μg/ml chloramphenicol, 10μg/ml tetracycline, and 1 mM DL-mevalonate. These seed cultures wereincubated at 37° C. until they reached a stationary growth phase. Thecell density of each seed culture was determined by measuring theoptical density of the culture at a wavelength of 600 nm (OD₆₀₀). Theseseed cultures were then used to inoculate 5 ml test cultures of LBmedium with appropriate antibiotics and increasing concentrations ofDL-mevalonate. The volume of seed culture used to inoculate each fresh 5ml culture was calculated to give an initial OD₆₀₀ value of 0.03. Testcultures were incubated for 48 hours at 30° C., after which growth wasarrested by chilling the cultures on ice. The optical density of eachculture was measured. One ml of each culture was harvested bycentrifugation (25000×g, 30 seconds), the supernatant was removed, andcell pellets were suspended in 500 μL of acetone by rapid mixing with aFisher Vortex Genie 2™ mixer (Scientific Industries, Inc., Bohemia,N.Y.). The acetone/cell mixtures were incubated at 55° C. for 10 minutesto aid in the extraction of lycopene from the cells. Extracted sampleswere centrifuged (25000×g, 7 minutes) to remove cell debris, and thecleared acetone supernatants were transferred to fresh tubes. Thelycopene concentration of each acetone extraction was assayed byabsorbance at 470 nm using a Beckman™ DU640 Spectrophotometer (BeckmanCoulter, Inc., Fullerton, Calif.) and a 400 μL quartz cuvette.Absorbance values at 470 nm were converted to lycopene concentrationsusing linear regressions from a standard curve produced using purelycopene (Sigma, St. Louis, Mo.). Final lycopene concentrations of eachstrain at increasing concentration of substrate is reported in FIG. 2.As shown in FIG. 2, lycopene production as a function of substrateconcentration following shaking for 48 hours at 30° C. demonstrated thatlycopene produced from natural levels of isopentenyl pyrophosphate innon-engineered Escherichia coli strain DH 10B (vertical stripes) remainsrelatively constant, while lycopene produced from isopentenylpyrophosphate generated by engineered Escherichia coli strain DPDXR1harboring the plasmid, pBBRMBI-2 (horizontal stripes), significantlyincreases at mevalonate substrate concentrations of 10 mM and higher.

EXAMPLE 4 Production of Carotenoids from Luria-Bertoni Broth Using theComplete Mevalonate Pathway

It was demonstrated that significantly higher levels of isopentenylpyrophosphate and isoprenoids derived therefrom were produced using thecomplete mevalonate-isoprenoid operon when compared to the native DXPpathway. The complete mevalonate-isoprenoid pathway was expressed usingthe two operons, MevT and MBI, which were expressed from pBADMevT andpBBRMBI-2, respectively, and coupled to pAC-LYC to demonstrate the invivo production of the carotenoid lycopene, using precursors produced byprimary cellular metabolism.

Escherichia coli strain DH10B was transformed with pBADMevT, pBBRMBI-2,and pAC-LYC by electroporation. Transformants were selected on LB agarplates containing 50 μg/ml carbenicillin, 10 μg/ml tetracycline, and 50μg/ml chloramphenicol. A single colony of the strain was transferredfrom the LB agar plate to 5 ml of LB liquid medium containing the sameantibiotics. This seed culture was incubated by shaking at 37° C. untilgrowth reached a stationary phase. The cell density of each seed culturewas measured at OD₆₀₀, and the cells were used to inoculate 5 ml testcultures of fresh LB medium plus the same antibiotics to give an OD₆₀₀of 0.03. The test cultures were incubated for 48 hours at 30° C., afterwhich growth was arrested by chilling the cultures on ice. The remainderof the experimental procedure was followed as described in Example 3.Final lycopene production (μg/ml lycopene per OD₆₀₀) of the pBADMevT,pBBRMBI-2, pAC-LYC plasmid system was compared to the lycopeneproduction from pAC-LYC plasmid only (control) in the Escherichia coliDH10B strain, as shown in FIG. 3. This figure illustrates, in graphform, the amount of lycopene produced for each strain, normalized forcell density, after shaking for 48 hours at 30° C. The column on theleft represents the amount of lycopene produced naturally in anon-engineered Escherichia coli strain (containing only pAC-LYC as acontrol). The column on the right represents the amount of lycopeneproduced from an Escherichia coli strain engineered to overproduceisopentenyl pyrophosphate from the mevalonate-isprenoid pathway.

EXAMPLE 5 Production of Terpenes by Coupling of Artificial MevalonateOperon(s) to Terpene Cyclases

Many valuable natural products were produced from the isoprenoidbiosynthetic pathways described herein. Depending on the desiredisoprenoid, the described operon(s) were modified, and/or additionaloperons or other means for chemical synthesis were provided to producethe precursors for the various classes. The following experimentsdemonstrated the synthesis of sesquiterpenes using the farnesylpyrophosphate synthase, ispA (SEQ ID NO 11), as well as the means bywhich other classes of isoprenoids, such as diterpenes, were synthesizedby varying the synthase cloned into the operon(s) to create the desiredprecursor.

In Vivo Production of Sesquiterpenes

Amorphadiene, a precursor to the antimalarial drug artemisinin, wasproduced from the co-expression of the mevalonate-isoprenoid pathway,along with a sesquiterpene cyclase-encoding amorphadiene synthesis. TheMBIS operon expressed from pBBRMBIS-2 was coupled withamorpha-4,11-diene synthase (ADS) for the in vivo production of thesesquiterpene amorpha-4,11-diene in Escherichia coli.

A gene coding for amorpha-4,1-diene synthase (ADS) was constructed sothat, upon translation, the amino acid sequence would be identical tothat described by Merke et al. (2000) Ach. Biochem. Biophys. 381(2):173-180. The ADS gene contains recognition sequences 5′ and 3′ of thecoding DNA corresponding to the restriction endonucleases NcoI and XmaI,respectively. The ADS gene was digested to completion with therestriction endonucleases, along with DNA for the plasmid pTrc99A. The1644-bp gene fragment and the 4155-bp plasmid fragment were purifiedusing 0.7% agarose gels and a Qiagen gel purification kit (Valencia,Calif.) according to the manufacturer's instructions. The two fragmentswere then ligated using T4 DNA ligase from New England Biolabs (Beverly,Mass.), resulting in plasmid pTRCADS. The insert was verified bysequencing to be the amorpha-4,11-diene synthase gene.

Escherichia Coli strain DH10B was transformed with both the pBBRMBIS-2and pTRCADS plasmids by electroporation. Bacterial colonies were thengrown on Luria-Bertoni (LB) agar containing 50 μg/ml carbenicillin and10 μg/ml tetracycline. A single bacterial colony was transferred fromthe agar plates to 5 ml LB liquid medium containing the same antibioticsand cultured by shaking at 37° C. for 16-18 hours. Five hundred μL ofthis culture was transferred into 5 ml fresh LB liquid medium with thesame antibiotics, then cultured by shaking at 37° C. to an opticaldensity of 0.816 at 600 nm (OD₆₀₀). A 1.6 ml portion of this culture wasused to inoculate a flask containing 100 ml of LB liquid medium with 50μg/ml carbenicillin and 10 μg/ml tetracycline, which was cultured byshaking at 37° C. After 1.5 hours, 1 ml of 1 M mevalonate and 100 μL of500 mM isopropylthio-β-D-galactoside (IPTG) were added to the culture,and it continued to be shaken at 37° C. Amorpha-4,11-diene concentrationwas determined by extracting 700 μl samples (taken hourly) with 700 μlof ethyl acetate in glass vials. The samples were then shaken at maximumspeed on a Fisher Vortex Genie 2™ mixer (Scientific Industries, Inc.,Bohemia, N.Y.) for three minutes. The samples were allowed to settle inorder to separate the ethyl acetate-water emulsions. Prior to gaschromatography-mass spectrometry analysis, the ethyl acetate layer wastransferred with a glass Pasteur pipette to a clean glass vial.

Ethyl acetate culture extracts were analyzed on a Hewlett-Packard 6890gas chromatograph/mass spectrometer (GC/MS). A 1 μl sample was separatedon the GC using a DB-5 column (available from, for example, AgilentTechnologies, Inc., Palo Alto, Calif.) and helium carrier gas. The ovencycle for each sample was 80° C. for two minutes, increasing temperatureat 30° C./minute to a temperature of 160° C., increasing temperature at3° C./min to 170° C., increasing temperature at 50° C./minute to 300°C., and a hold at 300° C. for two minutes. The resolved samples wereanalyzed by a Hewlett-Packard model 5973 mass selective detector thatmonitored ions 189 and 204 m/z. Previous mass spectra demonstrated thatthe amorpha-4,11-diene synthase product was amorphadiene and thatamorphadiene had a retention time of 7.9 minutes using this GC protocol.Since pure standards of amorpha-4,11-diene are not available, theconcentrations must be quantified in terms of caryophyllene equivalence.A standard curve for caryophyllene has been determined previously, basedon a pure standard from Sigma (St. Louis, Mo.). The amorpha-4,11-dieneconcentration is based on the relative abundance of 189 and 204 m/z ionsto the abundance of the total ions in the mass spectra of the twocompounds.

The amorphadiene concentration of the cultures seven hours after theaddition of IPTG and mevalonate is shown in FIG. 4. The figure shows theconcentration of amorphadiene produced seven hours after the addition ofmevalonate and isopropylthio-β-D-galactoside (IPTG). The column on theleft shows the concentration of amorphadiene produced fromnon-engineered Escherichia coli harboring the pTRCADS plasmid alone. Thecolumn on the right shows the concentration of amorphadiene producedfrom engineered Escherichia coli harboring the pBBRMBIS-2 and pTRCADSplasmids. The Escherichia coli strain engineered to make farnesylpyrophosphate from the mevalonate isoprenoid pathway produced 2.2 μg/mlamorphadiene, whereas the non-engineered strain (without the mevalonateisoprenoid pathway) produced only 0.13 μg/ml.

In Vivo Production of Diterpenes

The plasmid pBBRMBIS-2 was modified to include a gene encodinggeranylgeranyl pyrophosphate synthase (instead of farnesyl pyrophosphatesynthase). To demonstrate the utility of the artificialmevalonate-isoprenoid for in vivo diterpene production, this modifiedexpression system was coupled with a plasmid expressing casbenesynthase. Casbene synthase cDNA cloned into expression vector pET21-d(Hill et al. (1996), Arch Biochem. Biophys. 336:283-289) was cut outwith SalI (New England Biolabs, Beverly, Mass.) and NcoI (New EnglandBiolabs, Beverly, Mass.) and re-cloned into high-copy-number expressionvector pTrc99A. The gene fragment and the plasmid fragment were purifiedwith 0.7% agarose gels using a Qiagen gel purification kit (Valencia,Calif.) according to the manufacturers instructions. The two fragmentswere then ligated using T4 DNA ligase from New England Biolabs (Beverly,Mass.), resulting in plasmid pTrcCAS.

Escherichia coli strain DH10B was transformed with both the modifiedpBBRMBIS-2 and pTrcCAS plasmids by electroporation. Bacterial colonieswere then grown on Luria-Bertoni (LB) agar containing 50 μg/mlcarbenicillin and 10 μg/ml tetracycline. A single bacterial colony wastransferred from the agar plates to 5 ml LB liquid medium containing thesame antibiotics and cultured by shaking at 37° C. for 16-18 hours. Fivehundred microliters of this culture was transferred into 5 ml fresh LBliquid medium with 50 μg/ml carbenicillin and 10 μg/ml tetracycline, andcultured by shaking at 37° C. to an optical density of 0.816 at 600 nm(OD₆₀₀). A 150 μL portion of this culture was used to inoculate a flaskcontaining 25 ml of LB liquid medium with 50 μg/ml carbenicillin, 10μg/ml tetracycline, and 20 mM mevalonate. This mixture was cultured byshaking at 37° C. After 1.5 hours, 250 μL of 100 mM IPTG were added tothe culture, and it continued to be shaken at 37° C. Casbeneconcentration of the culture was determined hourly by extracting 450 μlsamples. To these samples was added 450 μL of ethyl acetate in a glassvial. The samples were then shaken on a Fisher Vortex Genie 2™ mixer(Scientific Industries, Inc., Bohemia, N.Y.) for three minutes. Thesamples were allowed to settle in order to separate the ethylacetate-water emulsion. The ethyl acetate layer was transferred with aglass Pasteur pipette to a clean vial.

Ethyl acetate culture extracts were analyzed on a Hewlett-Packard 6890gas chromatograph/mass spectrometer (GC/MS). A 1 μl sample was separatedon the CC using a DB-5 column (available from, for example, AgilentTechnologies, Inc., Palo Alto) and helium carrier gas. The oven cyclefor each sample was 80° C. for two minutes, increasing temperature at10° C./minute to a temperature of 300° C., and a hold at 300° C. for twominutes. The resolved samples were analyzed by a Hewlett-Packard model5973 mass selective detector that monitored ions 229, 257, and 272 m/z.Previous mass spectra had demonstrated that the casbene synthase productwas casbene and that casbene had a retention time of 16.6 minutes usingthis GC protocol. FIG. 5 shows the gas chromatographic analysis andresulting GC/MS chromatogram for the ethyl acetate extracts taken sevenhours after addition of IPTG from Escherichia coli engineered to produceisoprenoids from the artificial modified MBIS operon, thereby expressingthe casbene cyclase from the pTrcCAS plasmid. As a reference, FIG. 6shows the spectrogram for casbene.

1.-60. (canceled)
 61. A method for synthesizing mevalonate via amevalonate pathway in a host cell, wherein the method comprises: i)culturing a transformed host cell in a suitable medium, wherein thetransformed host cell is a prokaryote that does not normally synthesizeisopentenyl pyrophosphate (IPP) through the mevalonate pathway, andwherein the host cell comprises one or more nucleic acids heterologousto the host cell, wherein the one or more heterologous nucleic acidscomprises nucleotide sequences that encode two or more mevalonatepathway enzymes, wherein said two or more mevalonate pathway enzymescomprises an enzyme that condenses two molecules of acetyl-CoA toacetoacetyl-CoA and one or more additional mevalonate pathway enzymesselected from: (a) an enzyme that condenses acetoacetyl-CoA withacetyl-CoA to form HMG-CoA; and (b) an enzyme that converts HMG-CoA tomevalonate; said culturing providing for production of the two or moreenzymes, resulting in synthesis of said mevalonate by the transformedhost cell.
 62. The method of claim 61, wherein the one or moreheterologous nucleic acids is integrated into the chromosome of thetransformed host cell.
 63. The method of claim 61, wherein the one ormore heterologous nucleic acids is contained in at least oneextrachromosomal expression vector.
 64. The method of claim 61, whereinthe one or more heterologous nucleic acids is present in a singleexpression vector.
 65. The method of claim 61, wherein the enzyme thatcondenses two molecules of acetyl-CoA to acetoacetyl-CoA is anacetoacetyl-CoA thiolase.
 66. The method of claim 65, wherein thenucleotide sequence encoding the acetoacetyl-CoA thiolase comprises thenucleotide sequence set forth in SEQ ID NO:
 1. 67. The method of claim61, wherein the nucleotide sequence encoding the enzyme that condensesacetoacetyl-CoA with acetyl-CoA to form HMG-CoA comprises the nucleotidesequence set forth in SEQ ID NO:2.
 68. The method of claim 61 whereinthe nucleotide sequence encoding the enzyme that converts HMG-CoA tomevalonate comprises the nucleotide sequence set forth in SEQ ID NO:3.69. The method of claim 61, wherein the transformed host cell is of agenus selected from Escherichia, Enterobacter, Azotobacter, Erwinia,Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia,Shigella, Rhizobia, Vitreoscilla, and Paracoccus.
 70. The method ofclaim 61, wherein the transformed host cell is of the genus Escherichia.71. The method of claim 61, wherein the transformed host cell is anEscherichia coli.
 72. The method of claim 61, wherein the transformedhost cell is of the genus Enterobacter.
 73. The method of claim 61,wherein the transformed host cell is of the genus Azotobacter.
 74. Themethod of claim 61, wherein the transformed host cell is of the genusErwinia.
 75. The method of claim 61, wherein the transformed host cellis of the genus Bacillus.
 76. The method of claim 61, wherein thetransformed host cell is of the genus Pseudomonas.
 77. The method ofclaim 61, wherein the transformed host cell is of the genus Klebsiella.78. The method of claim 61, wherein the transformed host cell is of thegenus Proteus.
 79. The method of claim 61, wherein the transformed hostcell is of the genus Salmonella.
 80. The method of claim 61, wherein thetransformed host cell is of the genus Serratia.
 81. The method of claim61, wherein the transformed host cell is of the genus Shigella.
 82. Themethod of claim 61, wherein the transformed host cell is of the genusRhizobia.
 83. The method of claim 61, wherein the transformed host cellis of the genus Vitreoscilla.
 84. The method of claim 61, wherein thetransformed host cell is of the genus Paracoccus.
 85. A method forsynthesizing mevalonate via a mevalonate pathway in a host cell, whereinthe method comprises: i) culturing a transformed host cell in a suitablemedium, wherein the transformed host cell is Escherichia coli, andwherein the host cell comprises one or more nucleic acids heterologousto the host cell, wherein the one or more heterologous nucleic acidscomprises nucleotide sequences that encode two or more mevalonatepathway enzymes, wherein said two or more mevalonate pathway enzymescomprises an enzyme that condenses two molecules of acetyl-CoA toacetoacetyl-CoA and one or more additional mevalonate pathway enzymesselected from: (a) an enzyme that condenses acetoacetyl-CoA withacetyl-CoA to form HMG-CoA; and (b) an enzyme that converts HMG-CoA tomevalonate; said culturing providing for production of the two or moreenzymes, resulting in synthesis of said mevalonate by the transformedhost cell.
 86. The method of claim 85, wherein the one or moreheterologous nucleic acids is integrated into the chromosome of thetransformed host cell.
 87. The method of claim 85, wherein the one ormore heterologous nucleic acids is contained in at least oneextrachromosomal expression vector.
 88. The method of claim 85, whereinthe one or more heterologous nucleic acids is present in a singleexpression vector.
 89. The method of claim 85, wherein the enzyme thatcondenses two molecules of acetyl-CoA to acetoacetyl-CoA is anacetoacetyl-CoA thiolase.
 90. The method of claim 89, wherein thenucleotide sequence encoding the acetoacetyl-CoA thiolase comprises thenucleotide sequence set forth in SEQ ID NO:1.
 91. The method of claim85, wherein the nucleotide sequence encoding the enzyme that condensesacetoacetyl-CoA with acetyl-CoA to form HMG-CoA comprises the nucleotidesequence set forth in SEQ ID NO:2.
 92. The method of claim 85, whereinthe nucleotide sequence encoding the enzyme that converts HMG-CoA tomevalonate comprises the nucleotide sequence set forth in SEQ ID NO:3.